This invention relates to in vivo methods for characterizing interactions between molecules (e.g., protein and/or RNA molecules).
Numerous biologically important functions involve transient interactions between DNA molecules and proteins, RNA molecules and proteins, two or more proteins or RNA molecules, or ligands and receptors. For example, during most of the cell cycle, the tumor suppressor gene product pRb binds to the transcription factor E2F and represses its activity. E2F activity is provided by a family of at least seven proteins. The members of one subfamily (E2F-1, -2, -3, -4, and -5) form heterodimers with the members of another subfamily (DP-1 and -2). These heterodimers bind to the promoter of target genes and activate their transcription at certain stages of the cell cycle.
The transcriptional activity of the E2F/DP complexes can be repressed by any of several functionally related proteins termed the “pocket” proteins. Included in this category are proteins termed p107, p130, and pRb (the retinoblastoma protein). The pocket proteins exert their transcriptional inhibitory activity by directly interacting with the E2F/DP complexes. At the G1/S transition of the cell cycle, where E2F activity is required, the pocket proteins are phosphorylated which causes pRb and E2F to dissociate, leading to activation of the E2F transcription factor.
The physiological relevance of the interactions between E2F and the pocket proteins and between E2F and DP family members is supported by several observations: (i) in a variety of tumors, both copies of the RB gene contain loss of function mutations, and reintroduction of the wild-type RB gene reduces tumorigenicity; (ii) overexpression of E2F-1 in an experimental system can lead to neoplastic transformation; (iii) PRAD1, the gene which encodes cyclin D, a positive regulatory subunit of the pRb kinases, is, as the result of a chromosomal rearrangement, overexpressed in numerous tumors; (iv) disruption of the interaction of E2F with proteins is required for the oncogenic activity of certain DNA tumor viruses. oncogenic proteins such as E1A of adenoviruses, the large T antigen of SV40, and E7 of Human Papilloma Viruses can abrogate pRb-mediated repression of E2F, causing the host cell to enter the cell cycle inappropriately. Compounds which can destabilize the interaction of an oncogenic viral protein with pRb without affecting the interaction of pRb with E2F can be used therapeutically to treat or prevent cancers associated with these viruses.
Previous studies of interactions between regulatory proteins have revealed important paradigms about how proteins interact with each other. For example, studies of protein/protein interactions have led to the identification of several structural motifs (e.g., the helix-loop-helix motif, SH2 and SH3 domains, and the leucine zipper). The primary amino acid sequences of E2Fs, DPs, and the pocket proteins do not resemble any of the known motifs. Thus, a convenient method which permits a detailed study of the protein/protein interactions involved in this novel family of regulatory proteins may reveal new motifs for protein/protein interactions. The E2F-1/DP-1 interaction domain has been mapped to amino acids 120–310 of E2F-1 and amino acids 205–277 of DP-1. In contrast, the E2F-1/pRb interaction domain has been mapped to amino acids 409–427 of E2F-1. Thus, the DP-1 and pRb binding sites on E2F-1 do not overlap. Accordingly, certain mutations may affect the ability of E2F-1 to bind to DP-1 without affecting the ability of E2F-1 to bind to pRb. Similarly, certain compounds may affect the ability of E2F-1 to bind to DP-1 without affecting its ability to bind to pRb.
Counterselectable Markers: While selectable markers have been used to, under certain conditions, promote the growth of only those cells which express the selectable markers, counterselectable marker have been used, under certain conditions, to promote the growth of only those cells which have lost the counterselectable marker. Counterselectable markers when present on plasmids can be used to select for cells that have lost the plasmid, a process called plasmid “shuffling” (see, e.g., Sikorski and Boeke, 1991, Meth. in Enzymol. 194:302). For example, expression of the URA3 gene, which encodes orotidine-5′-phosphate, is lethal in the presence of a medium containing 5-fluoro-orotic acid (5-FOA). Cells expressing URA3 can also be positively selected for by growing them on uracil-free media; thus, depending on the growth conditions, URA3 can be used either for positive or negative conditions. The LYS2 gene, which encodes α-aminoadipate reductase, can also be used for counterselection; yeast cells which express LYS2 will not grow on a medium containing α-aminoadipate as a primary nitrogen source. Similarly, expression of LYS5 on a medium containing α-aminoadipate is lethal. These genes, which are involved in lysine biosynthesis, can be selected in a positive fashion on a lysine-free medium. Another counterselectable reporter gene is the CAN1 gene which encodes an arginine permease. Expression of this gene in the absence of arginine and in the presence of canavanine is lethal. Similarly, expression of the counterselectable gene CYH2 is lethal in the presence of cycloheximide. Expression of a counterselectable reporter gene has been used to identify mutations in the activation domain of estrogen receptor which inhibit its ability to activate transcription (Pierrat et al., 1992, Gene 119:237–245).